More Notes on PCR
I am going to attempt to get the rest of what I've learned on PCR in my notebook today,to have it all neatly organized in one place:
- DNA templates denature depending on their quantity of (G+C), guanine and cytosine. The higher their concentration, the higher a temperature is needed to denature the DNA. Also longer strands need more time to denature, and they start detaturing in places that are rich in thymine and adenine. With Taq, the suggest time and temperature are 45 sec. and 94-95°C.
- The annealing temperature needs to be very carefully selected. If it is too high, the DNA will anneal poorly, while at a low temperature then nonspecific annealing can occur. Typically this needs to be ~3-5°C lower than the melting point of the primers, but it is probably a good idea to optimize it depending on your specific primers.
- During the extension phase the temperature should be at 72-78°C, and should last for about a minute per thousand base pairs in the template DNA, with the last cycle lasting up to three times as long as a normal cycle.
I will go over the specific procedure, and the amount of reagents we are using tomorrow.